Diagnostic test kit for determining a predisposition for breast and ovarian cancer, materials and methods for such determination

ABSTRACT

The present invention relates generally to the field of human genetics, and more specifically to the detection of a specific type of germline mutations in the BRCA1 gene, which will predispose to breast and ovarian cancer. In addition, the invention relates to the molecular genetic mechanism that may have mediated the genesis of these mutations, in particular the role of Alu repetitive DNA elements present in the intronic regions of BRCA1. The invention further relates to somatic mutations of this type in the BRCA1 gene in human breast and ovarian cancer, and their use in the diagnosis and prognosis of human breast and ovarian cancer. The invention more particularly relates to the screening of this type of BRCA1 mutations in human genomic DNA, which are useful for the diagnosis of inherited predisposition to breast and ovarian cancer.

The present invention relates generally to the field of human genetics.In particular the invention relates to methods and means (diagnostictest kits) for studying the predisposition for certain types of cancersoften having a hereditary component and more specifically to thedetection of a specific type of germline mutations in genes involved orassociated with certain types of hereditary cancers, in particular the(human) BRCA1 gene, which will predispose to breast and ovarian cancer.In addition, the invention reveals a molecular genetic mechanism thatmay have mediated the genesis of these mutations, in particular the roleof Alu repetitive DNA elements present in the intronic regions of BRCA1.The invention further relates to somatic mutations of this type in theBRCA1 gene in human breast and ovarian cancer, and their use in thediagnosis and prognosis of human breast and ovarian cancer.

The invention also relates to the screening of this type of BRCA1mutations in human genomic DNA, as part of clinical protocols for thediagnosis of inherited predisposition to breast and ovarian cancer.

BACKGROUND OF THE INVENTION

Breast cancer is the most common malignancy among women in theNetherlands, with a cumulative risk by age 85 of one in 11. Thestrongest epidemiological risk factor for the disease is a positivefamily history.

Depending on the age of diagnosis and occurrence of bilateral disease inthe index case, first degree relatives may have a relative risk of up to10 for developing breast cancer. In the US population, 6 to 19% of womenwith breast cancer have at least one affected relative at the time ofdiagnosis [11], but not all of them are expected to be true geneticcases as the high incidence of breast cancer in the general populationwill inevitably cause some coincidental familial clustering. In anattempt to stratify the two classes, criteria to define truly inheritedbreast cancer have been proposed [2]. Such cases are characterized byearly age of onset (premenopausal), excess of bilaterality, and clearpaternal or maternal transmission with an autosomal dominant mode ofinheritance. Approximately 5% of all cases comply with these criteria,while another 13% are classified as familial clustering [3]. Since earlyage of onset appears to be a hallmark of hereditary breast cancer, onemay suspect that among these cases the genetic component is much higher.Indeed, up to 36% of cases diagnosed under the age of 30 are expected tobe genetic [4]. No such data are available for the Dutch situation, andlittle or none of this has been confirmed at the molecular geneticlevel.

Linkage analysis of early-onset breast cancer families localized BRCA1to the long arm of chromosome 17 [5]. Further analyses of additionalfamilies revealed that women inheriting a mutant allele of BRCA1 arealso at increased risk for ovarian cancer [6,7]. Overall, approximately45% of all families in which breast cancer is the predominant malignancyare due to BRCA1, as are over 80% of all families with both breast andovarian cancer [6,8]. Female mutation carriers have been estimated tohave an 87% risk to develop breast cancer before the age of 70, and 63%risk to develop ovarian cancer before that age [7]. However, significantevidence for ovarian cancer risk heterogeneity was obtained, indicatingthe existence of at least two classes of BRCA1 mutations; one conferringa high risk to both breast and ovarian cancer, and one conferring a highrisk to breast cancer, but only a moderate risk to ovarian cancer, withthe former comprising approximately 26% of all BRCA1 mutations [9]. Thegene frequency of BRCA1 has been estimated to be 1 in 833 women [10].This would imply that 1.7% of all breast cancer patients diagnosedbetween age 20 and 70 are carrier of such a mutation.

The gene structure of BRCA1 was found to consist of 22 coding exonsspanning >80 kb of genomic DNA [11], and encoding a 7.8 kb transcript[12]. An unusually large exon 11 of 3.4 kb comprises 61% of the codingdomain. Over 900 mutations in BRCA1 have been published to date andcompiled into an electronically accessible database [13]. Severalcharacteristics stand out [14]. First, they are nearly ubiquitouslydistributed over the gene. Second, >85% of the mutations in the databaselead to premature termination of protein translation. These includebasepair substitutions leading to a stop codon, small insertions anddeletions (of 1 to 40 basepairs) leading to a frame-shift, orsplice-site mutations leading to deletions of complete exons and frameshifts. That these changes presumably inactivate gene function issupported by the finding that the great majority of breast and ovariantumours that develop in BRCA1 mutation carriers show loss of thewildtype allele [15]. The relevance in terms of cancer predisposition ofthe missense mutations remains a matter of debate. Some of them appearrare polymorphic variants, as they are also observed in control samples.Others seem to affect critical residues, such as the cysteines in theamino-terminal ring finger domain [12], which are conserved in the mouseBrca1 sequence [16]. Third, a number of mutations have been foundrepeatedly, reducing the number of distinct mutations to about 150. Twoof these, the 185delAG mutation and the 5382insC mutation, eachrepresent approximately 11% of all mutations thus far reported [14].Reconstruction of the haplotypes bearing some of the most commonmutations has provided strong evidence that they have either a single ora few common ancestors and may have been present in the populationalready for several centuries [17-19]. Consequently, the incidence ofspecific mutations is strongly dependent on the population from whichthe breast cancer families were ascertained. Thus the 185delAG mutationwas picked up mainly in families of Ashkenazi-Jewish origin [20].

The extent of the founder-effect was highlighted by the finding thatapproximately 1% of all Ashkenazi Jews (i.e. regardless of a positivebreast cancer family history) are carrying this mutation [21,22], 8times that of the incidence of all mutations together in the generalpopulation [10]. Specific mutations have also been recurrently detectedin breast cancer families of Swedish, British, Italian, and Austrianorigin [18,23-26].

Despite the vast number of BRCA1 gene changes detected to date, thereremains a discrepancy between the proportion of BRCA1 mutationspredicted by linkage studies [6,8], and the actual prevalenceestablished by mutation analysis, among breast cancer families derivedfrom a variety of ethnic backgrounds [27-31]. In general, this isexplained in two ways: either a substantial number of mutations havebeen missed by the applied mutation screening methodology, or thegenetic heterogeneity of hereditary breast cancer is significantlygreater than hitherto expected.

Relatively little information of predictive value can be gleaned fromthe existing data. In one set of 35 kindreds with proven BRCA1 mutationsfrom the United Kingdom, the ovarian cancer risk heterogeneity aspredicted from linkage studies could be confirmed [25]. Mutationsoccurring before codon 1435 conferred a significantly higher ovariancancer risk than those occurring after this point. While this isconsistent with earlier predictions based on linkage analysis [9], thecurrent mutation distribution is at odds with the predicted lowerfrequency of these alleles. In addition, the expressivity of BRCA1displays considerable inter-family variability. For example, the185delAG mutation was detected in families with early-onset breastcancer and ovarian cancer, or late-onset breast cancer without ovariancancer [32]. Clearly, other factors influence the expression of thephenotype, and some of those might be genetic, others environmental. Ofnote, BRCA1 carriers who have a rare allele at the HRAS1 minisatellitelocus were recently shown to be at a 2.8-fold increased risk for ovariancancer relative to those carriers who had common alleles at HRAS1 [33].However, a firm establishment of the full spectrum of BRCA1 gene changesin the population is pivotal for a more formal analysis of this matter.

An intriguing feature of BRCA1, and unexpected in the light of Knudson'stwo-hit inactivation theorem for tumor suppressor genes, is thatsomatically acquired mutations are extremely rare in ovarian tumors[34-38] and have in fact not yet been detected in 135 breast tumors[39-40]. This might indicate that inactivation of BRCA1 is not selectedfor during tumorigenesis of the non-inherited form of breast cancer.BRCA1 expression might be critical only during certain stages of tissuedevelopment, e.g., during puberty when the breast undergoes its finaldifferentiation into a potential milk-producing gland [39]. However,others have argued that the mechanism of inactivation might be differentfrom that seen in inherited cases [41].

SUMMARY OF THE INVENTION

The present invention now reveals that the unusual high concentration ofAlu-elements in the BRCA1 gene intronic regions [11] favors theinduction of large genomic deletions and inversions in a situation ofincreased genomic instability although other mechanisms leading to thesemutations may also play significant roles. The present invention thusprovides a diagnostic test kit (and means and methods) for determiningmutations, especially deletions of relatively large stretches ofnucleotides in genes associated with hereditary types of cancer, inparticular such mutations (deletions of relatively large stretches ofnucleotides) in the BRCA1 gene. Such mutations are difficult, if notimpossible, to detect by the current PCR-based approach (if theiroccurrence or the site thereof is unknown) using genomic DNA astemplate, which has been most widely applied to establish the currentmutation spectrum of BRCA1.

The present invention thus provides a diagnostic test kit for detectingthe presence of or predisposition for e.g. breast cancer, whereby ameans is provided for detecting a deletion of a stretch of nucleotidesfrom a BRCA 1 gene in a sample. Now that it is known that such mutationsoccur, it is within the skill of the art to arrive at means to determinethe presence of these mutations, either the ones disclosed herein orsimilar mutations. Such means may include hybridization of a probeflanking both sides of the deletion, or using two probes on either sideof the deletion and amplifying the stretch in between, another way maybe lack of hybridization, when using a probe hybridizing to a deletedpart, etc. Yet another way may be lack of amplification between one ormore sets of primers targeted at or near a deleted region. This alreadyimplicates that typically multiplex PCR approaches are very suitable.Also exon-connection PCR is a very suitable approach for use in thepresent invention. The techniques mentioned above are well known in theart and need no further explanation. Since mutations as disclosed hereinmay occur in one allele only, quantitative methods are often preferable.It is of course clear that the diagnostic test kit should provide allother necessary means for determining the presence or absence of themutations, such as buffers, detection means (possibly labels ormarkers), etc.

A convenient diagnostic test kit according to the invention apart fromamplification methods such as PCR, NASBA and the like is a diagnostictest kit whereby the means comprise the necessary elements for southernblotting. The deletions to be detected are typically relatively largestretches of nucleotides, particularly of a size which when subjected toPCR or similar amplification techniques would not be amplified undernormal reaction conditions because of their length. Typically thedeletion comprises one or more exons of the BRCA1 gene or a frameshiftand/or a termination codon. An exemplified deletion that is a goodmarker for the predisposition for cancer is the deletion which comprisesat least a major part of exon 22.

Another exemplified deletion that is a good marker for thepredisposition for cancer is the deletion which comprises at least amajor part of nucleotides 1396-1662.

Another exemplified deletion that is a good marker for thepredisposition for cancer is the deletion which comprises at least amajor part of exons 13-16.

Another exemplified deletion that is a good marker for thepredisposition for cancer is the deletion which comprises at least amajor part of exon 13.

An exemplified deletion that is a good marker for the predisposition forcancer is the deletion which comprises a stretch of nucleotides betweentwo ALU-elements. This kind of deletion ties in very nicely with asuggested mechanism of the origin of these mutations and the same mayalso be found in other genes involved in cancer and having many of theseelements.

Thus the invention further provides a probe for use in a diagnostic testkit according to invention comprising a nucleic acid sequence which is afusion of two (complementary sequences of) ALU elements, in particularof the BRCA1 gene. In general the invention thus provides a probe foruse in a diagnostic test kit according to the invention, which is afusion product of two sequences adjacent to the site of a deletion of astretch of nucleotides.

Also provided is a method for determining the presence in a sample of anucleic acid derived from a BRCA1 gene having a deletion of a stretch ofnucleotides, comprising contacting said sample with at least one probewhich alone or together with other means is capable of distinguishingbetween BRCA1 genes having said deletion and BRCA1 genes not having saiddeletion, allowing for possible hybridization between said probe andsaid nucleic acid and identifying the hybridization product.

Specific embodiments of the invention will be explained in detail below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention in one of its embodiments, which has beendescribed in detail in the experimental part provides a description anddetection in human genomic DNA of large genomic deletions in BRCA1. Inaddition, the invention shows involvement of the Alu-repeat elements,present at high frequency in the intronic regions of BRCA1 [11], ingenerating a number of these deletions. The invention also contemplatesthe frequency of these deletions in the Dutch population, and theirdescendance from a common ancestor.

We have found that the mutation spectrum of BRCA1 as resolved up to thispoint. [13,42] has been biased by PCR-based mutation-screening methodssuch as SSCP, the protein truncation test (PTT), and direct sequencing,using genomic DNA as template. We describe as examples thereof two largegenomic deletions, which are not detected by these approaches, and whichtogether comprise 38% of all BRCA1 mutations found in a sample of 170Dutch breast cancer families [43,44]. One deletion removes 510 basepairs(bp) including exon 22 (FIG. 1) and was found 8 times. The otherdeletion removes 3835 bp including exon 13 (FIG. 2) and was found 4times.

The haplotypes of the 8 families with the exon 22 deletion werereconstructed by typing 3 intragenic markers (D17S855, D17S1322,D17S1323) and 2 flanking markers (THRA1 and D17S1327). These haplotypeswere completely concordant for the intragenic markers in at least 7families, and the haplotype conservation extended proximally to THRA1,and distally to D17S1327, in at least 5 families, to comprise a geneticregion of approximately 2 cM. The haplotypes of the 4 families with theexon 13 deletion were reconstructed in a similar way. These haplotypeswere completely concordant for the intragenic markers in at least 2families, and the haplotype conservation extended proximally to THRA1,and distally to D17S1327, in all 4 families, to comprise a geneticregion of approximately 2 cM.

Molecular characterization of the deletions revealed that the exon 22deletion starts in intron 21 and ends within the most upstream copy ofthree head-to-tail arranged Alu-elements in intron 22. A 17-bp imperfecthomology to the intron 22 Alu-element was found at the 5′ deletionbreakpoint (FIG. 3). The 3′ breakpoint is closely flanked on either sideby two 25-bp sequences strongly homologous to the Alu core-sequenceimplied to stimulate recombination [45].

The exon 13 deletion starts in intron 12 in an Alu-element (112 bp fromthe 5′ end) and ends in intron 13 in a region which shares very highhomology to this element (FIG. 4). Both the 5′ and the 3′ breakpoint areclosely flanked on either side by sequences strongly homologous to the26-bp Alu core-sequence implied to stimulate recombination [45].

The current invention facilitates the design of PCR-based strategies(now that the presence of this kind of mutations is known) to identifythe heterozygous presence of the deletions in human genomic DNA.Oligonucleotide primers can be designed so to immediately flank thedeletion breakpoints, and allow the specific amplification of adeletion-junction fragment as a diagnostic endpoint. Given the size ofthe deletions, the wildtype BRCA1 genomic sequence would remainrefractory to PCR-amplification under most standard reaction conditions.PCR-based diagnosis is an essential requirement to scale up throughputin the screening for these mutations.

The current invention also pertains to the molecular mechanism which mayhave generated the genomic deletions in the BRCA1 gene, especially sincethis needs to be viewed in a broader sense in that the same kind ofphenomenon may be picked up in other genes or in the same gene, but nothaving anything to do with the inheriting kind of cancer.

The current invention thus also pertains to the role of BRCA1 mutationsin non-inherited or sporadic breast cancer.

EXAMPLES

The exon 22 deletion was revealed by Southern blot analysis of genomicDNA digested with either HindIII or BglII. As probe we used p1424, whichcontains ^(˜)1-kb cDNA-deletion segment from exons 14-24. A carrier ofthe exon 22 deletion shows aberrant bands of 9.3 kb in the HindIIIdigest and of 6.7 kb in the BglII digest.

The exon 13 deletion was revealed by Southern blot analysis of genomicDNA digested with either HindIII or BglII. As probes we used either p11or p1424, which contain ^(˜)1-kb cDNA-derived segments from exon 11 andexons 14-24, respectively. A carrier of the exon 13 deletion shows anaberrant band of 6.4 kb in the HindIII pattern obtained with probe p1424and of 14 kb in the BglII pattern obtained with probe p11.

To further characterize these deletions, we used intronic amplimers toobtain PCR-products from genomic DNA, specifically containing thedeletion-junction fragment. Amplimers flanking exon 22 generated anaberrant genomic fragment of 1.4 kb in DNA samples carrying the exon 22deletion, which turned out to contain a 510-bp deletion relative to thewildtype sequence (FIG. 3). The deletion affecting exon 22 removes thebases 79505-80014 (510 bp) as listed in the genomic sequence of BRCA1(Genbank accession nr. L78833). As a result, 74 basepairs, correspondingto exon 22, are missing in the processed mRNA-transcript (bases79543-79616 in Genbank accession nr. L78833).

Amplimers flanking exon 13 generated an aberrant genomic fragment of 2.7kb in DNA samples carrying the exon 13 deletion, which turned out tocontain a 3835 bp deletion relative to the wildtype sequence (FIG. 4).The deletion affecting exon 13 removes the bases 44514-48348 (3835basepairs) as listed in the genomic sequence of BRCA1 (Genbank accessionnr. L78833). As a result, 172 basepairs, corresponding to exon 13, aremissing in the processed mRNA-transcript (nucleotides 46156-46327 inGenbank accession nr. L78833).

We examined 142 breast cancer families in which thusfar no BRCA1 orBRCA2 mutation had been found (refs. 43,44 and our unpublished results)for the presence of the exon 13 and exon 22 deletions. They were foundin 4 and 8 families, respectively. Together with previous mutationscreening results, using PTT and direct sequencing [44], these deletionsthus comprise {fraction (12/32)} (38%) of all families in which a BRCA1mutation has been detected to date. Three intragenic and 2 flankingmarkers were used to reconstruct the disease haplotype for each of theresearch families carrying either the 510-bp or 3.8-kb deletion. Strongconservation of allele-lengths was observed at the intragenic loci amongthe haplotypes carrying the same deletion, in agreement with theirdescent from a common ancestor.

The haplotype in the Dutch population that carries the 510-bp deletionaround exon 22 is characterized by a 155-bp allele at the microsatellitemarker D17S855 in intron 20, a 122-bp allele at microsatellite markerD17S1322 in intron 19, and a 151-bp allele at microsatellite markerD17S1323 in intron 12. The haplotype in the Dutch population thatcarries the 3835-bp deletion around exon 13 is characterized by a 151-bpallele at D17S855, a 122-bp allele at D17S1322, and a 151-bp allele atD17S1323 in intron 12. The primer sequences used to detect these allelesare: for D17S1322: Foward (F) 5′CTAGCCTGGGCAACAAACGA 3′ (SEQ ID NO:1)and Reverse (R) 5′ GCAGGAAGCAGGAATGGAAC 3′ (SEQ ID NO:2); for D17S855: F5′ GGATGGCCTTTTAGAAAGTGG 3′ (SEQ ID NO:3) and R 5′ ACACAGACTTGTCCTACTGC3′ (SEQ ID NO:4); for D17S1323: F 5′ TAGGAGATGGATTATTGGTG 3′ (SEQ IDNO:5), and R 5′ AAGCAACTTTGCAATGAGTG 3′ (SEQ ID NO:6). PCR conditionshave been described elsewhere [44].

Detection of the Mutations

Isolation of genomic DNA and total RNA from freshly taken blood samples,and preparation of first-strand cDNA by reverse transcription, has beendescribed [43].

cDNA Analysis to Detect the Exon 13 Deletion

Exons 12-24 were amplified from first-strand cDNA products obtained byreverse transcription using the following primers for the first PCR: F5′TCACAGTGCAGTGAATTGGAAG 3′ (SEQ ID NO:7) and R 5′GTAGCCAGGACAGTAGAAGGACTG (SEQ ID NO:8) 3′. The obtained PCR-productswere used as template for a second PCR of exons 12-24 using nestedprimers (F 5′ GAAGAAAGAGGAACGGGCTTGG 3′ (SEQ ID NO:9) and R 5′GGCCACTTTGTAAGCTCATTC 3′ (SEQ ID NO:10)). PCR conditions were asdescribed previously [43]. Five μl of the final PCR products areanalysed on a 1% agarose gel.

cDNA Analysis to Detect the Exon 22 Deletion

Exons 12-24 were amplified from first-strand cDNA products obtained byreverse transcription using the following primers for the first PCR: F5′TCACAGTGCAGTGAATTGGAAG 3′ (SEQ ID NO:7) and R 5′GTAGCCAGGACAGTAGAAGGACTG 3′ (SEQ ID NO:8). The obtained PCR-productswere used as template for a second PCR of exons 20-24 using nestedprimers (F 5′ AACCACCAAGGTCCAAAGC 3′ (SEQ ID NO:11) and R 5′GTAGCCAGGACAGTAGAAGGACTG 3′ (SEQ ID NO:12)). PCR conditions were asdescribed previously [43]. Five μl of the final PCR products areanalysed on a 1% agarose gel.

Genomic PCR of the 3835-bp deletion spanning exon 13. A PCR reaction of50 μl contains 200 ng of genomic DNA, 10 pmol primers (F 5′TAGGAGATGGATTATTGGTG 3′ (SEQ ID NO:5) and R 5′ TACGTGCGTTCAACTGAAGC 3′(SEQ ID NO:13)), 0.75 Units Amplitaq Taq polymerase(Perkin-Elmer-Cetus), and 5 μl of 10×ITP/BSA buffer (500 mM KCl, 100 mMTRIS-HCl pH 8.4, 25 mM MgCl₂, 2 mg/ml BSA, 2 mM dNTPs). This mixture isheated at 94° C. for 5 minutes, followed by 35 cycles of PCR (at 94° C.for 45 seconds, at 52° C. for 1 min. and at 72° C. for 2.5 min on aPerkin-Elmer-Cetus DNA thermal Cycler). The PCR is concluded by anincubation at 72° C. for 6 minutes. Five μl of the PCR products areanalysed on a 1% agarose gel.

Genomic PCR of the 510-bp deletion spanning exon 22. A PCR reaction of50 μl contains 300 ng of genomic DNA, 10 pmol primers (F 5′TCCCATTGAGAGGTCTTGCT 3′ (SEQ ID NO:14) and R 5′ ACTGTGCTACTCAAGCACCA 3′(SEQ ID NO:15)), 0.75 U Amplitaq Taq polymerase (Perkin-Elmer-Cetus), 5μl Optiprime buffer #6 (Stratagene) and 0.1 mM dNTPs. Thermal cycles areas described for the deletion of 3.8 kb. Five μl of the PCR products areanalysed on a 1.5% agarose gel.

Southern Analysis

Five μg of genomic DNA is digested with either the restrictionendonuclease BglII or HindIII. Agarose gels (0.8%) are run at 30V for 16hr in TAE buffer (40 mM Tris-HAc pH 8.3, 1 mM EDTA). Procedures fordenaturing, and transferring the separated DNA to nylon membranes(Hybond N+, Amersham) have been described [46]. As probes we usedPCR-products obtained from a clone containing the complete BRCA1-cDNA,and purified by using the QIAquick PCR Purification Kit from QIAGEN.Probe-11 (p11) derives entirely from exon 11 and was obtained with theprimers F 5′ GAAAAAAAAGTACAACCAAATGCC 3′ (SEQ ID NO:16) and R 5′AGCCCACTTCATTAGTACTGGAAC 3′ (SEQ ID NO:17), and probe-1424 (p1424)contains exons 14-24 and was obtained with the primers F 5′TACCCTATAAGCCAGAATCCAGAA 3′ (SEQ ID NO:18) and R 5′ GGCCACTTTGTAAGCTCATTC 3′ (SEQ ID NO:19). Purified fragments were labelled using theMegaprime DNA labelling System from Amersham according to suppliersprotocols. Hybridizations were carried out at 65° C. in 125 mMNa2HPO4.2H2O, 7% SDS, 10% PEG-6000, 1 mM EDTA. Final washing was in 45mM NaCl, 4.5 mM Na-citrate pH 7.0, 0.1% SDS, at 65° C. for 30 minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the genomic deletion spanning exon22 of BRCA1. The intronic regions are drawn to scale relative to oneanother, and the exonic region are drawn to scale relative to oneanother, but not to intronic regions. The positions of the restrictionendonucleases HindIII and BglII, used in Southern blot analysis, areindicated. The arrows indicate the presence and orientation of anAlu-element.

FIG. 2. Sequence of exon 22 (upper case) and its flankingintron-sequence (lower case) (SEQ ID NO:20). The numbers refer to thegenomic sequence of BRCA1 (Genbank accession nr. L78833). Starting andending positions of the 510-bp deletion are indicated by hooked arrowsand affect positions 79505-80014. The first 241 bp of an Alu-element aredepicted in italics, and the boxed sequences are imperfect copies (1 and5 mismatches, respectively) of a common 26-bp core sequence involved inrecombinations leading to gene rearrangements in the LDLR gene [45]. Astretch of 17 bp at the 5′ junction of the deletion is homologous to a19-bp stretch 37 bp upstream of the 3′ deletion-breakpoint (underlinedwith arrows).

FIG. 3. Schematic representation of the genomic deletion spanning exon13 of BRCA1. The intronic regions are drawn to scale relative to oneanother, and the exonic region are drawn to scale relative to oneanother, but not to intronic regions. The positions of the restrictionendonucleases HindIII and BglII, used in Southern blot analysis, areindicated. The arrowheads indicate the presence and orientation of anAlu-element.

FIG. 4. Aligned sequences of intronic regions flanking exon 13 (SEQ IDNOS.21-22), and of the deletion-junction fragment (Jnctn) (SEQ IDNO:23). The upper sequence of each alignment corresponds to intron 12sequences, (SEQ ID NO:21) the lower sequence intron 13 sequences (SEQ IDNO.22). The numbers refer to the genomic sequence of BRCA1 (Genbankaccession nr. L78833). The boxed sequence indicates the 10 bp where therecombination took place that led to the deletion of 3835 bp. The intron12 sequence depicted here represents the first 180 bp of an Alu-element.The intron 12 region 44481-44551 shares an 85% identity with the intron13 region 48316-48386. The underlined sequences are imperfect copies ofa common 26-bp core sequence involved in recombinations leading to generearrangements in the LDLR gene [45].

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23 1 20 DNA Artificial Sequence Description of Artificial Sequenceprimer forward for D17S1322 1 ctagcctggg caacaaacga 20 2 20 DNAArtificial Sequence Description of Artificial Sequence primer reversefor D17S1322 2 gcaggaagca ggaatggaac 20 3 21 DNA Artificial SequenceDescription of Artificial Sequence primer forward for D17S855 3ggatggcctt ttagaaagtg g 21 4 20 DNA Artificial Sequence Description ofArtificial Sequence primer reverse for D17S855 4 acacagactt gtcctactgc20 5 20 DNA Artificial Sequence Description of Artificial Sequenceprimer forward for D17S1323 5 taggagatgg attattggtg 20 6 20 DNAArtificial Sequence Description of Artificial Sequence primer reversefor D17S1323 6 aagcaacttt gcaatgagtg 20 7 22 DNA Artificial SequenceDescription of Artificial Sequence primer forward for first PCR 7tcacagtgca gtgaattgga ag 22 8 24 DNA Artificial Sequence Description ofArtificial Sequence primer reverse for first PCR 8 gtagccagga cagtagaaggactg 24 9 22 DNA Artificial Sequence Description of Artificial Sequenceprimer forward for second PCR 9 gaagaaagag gaacgggctt gg 22 10 21 DNAArtificial Sequence Description of Artificial Sequence primer reversefor second PCR 10 ggccactttg taagctcatt c 21 11 19 DNA ArtificialSequence Description of Artificial Sequence primer forward 11 aaccaccaaggtccaaagc 19 12 24 DNA Artificial Sequence Description of ArtificialSequence primer reverse 12 gtagccagga cagtagaagg actg 24 13 20 DNAArtificial Sequence Description of Artificial Sequence primer reverse 13tacgtgggtt caactgaagc 20 14 20 DNA Artificial Sequence Description ofArtificial Sequence primer forward 14 tcccattgag aggtcttgct 20 15 20 DNAArtificial Sequence Description of Artificial Sequence primer reverse 15actgtgctac tcaagcacca 20 16 24 DNA Artificial Sequence Description ofArtificial Sequence primer forward 16 gaaaaaaaag tacaaccaaa tgcc 24 1724 DNA Artificial Sequence Description of Artificial Sequence primerreverse 17 agcccacttc attagtactg gaac 24 18 24 DNA Artificial SequenceDescription of Artificial Sequence primer forward 18 taccctataagccagaatcc agaa 24 19 21 DNA Artificial Sequence Description ofArtificial Sequence primer reverse 19 ggccactttg taagctcatt c 21 20 720DNA Homo sapiens /note=“Exon 22 of BRCA1 and its flanking intronsequences, pos. 79441-80160” 20 agaggtcttg ctataagcct tcatccggagagtgtagggt agagggcctg ggttaagtat 60 gcagattact gcagtgattt tacatctaaatgtccatttt agatcaactg gaatggatgg 120 tacagctgtg tggtgcttct gtggtgaaggagctttcatc attcaccctt ggcacagtaa 180 gtattgggtg ccctgtcaga gagggaggacacaatattct ctcctgtgag caagactggc 240 acctgtcagt ccctatggat gcccctactgtagcctcaga agtcttctct gcccacatac 300 ctgtgccaaa agactccatc tgtaagggatgggtaaggat ttgagaactg cacatattaa 360 atatactgag ggaagacttt ttccctctaactctttttcc catatgtccc tccccctcct 420 ctctgtgact gccccagcat actgtgtttcaacaaatcat caagaaatga tgggctggag 480 gctgggcatg gtggctcatg tctgtaatcccagcactttg ggaggccgag gcaggtggat 540 cacttgtcag gagtttgaga ccagcctggccaacatggtg aaaccccatc tgtactaaaa 600 aaaaaaaaac aaaaagtagc caggcctggtggagcatgcc tgtaatgcca gctatttggg 660 aagttgaggt gtgagcatcg cttgaacgtgggaggcagag gttgcagtga gccaagattg 72 21 178 DNA Homo sapiens/note=“Intronic region flanking exon 12, pos. 44423 - 44600” 21cctgtaatcc cagcactttg ggaggccgag gcgggaggat catgtggtca ggagatccag 60accatcctgg ctaacacggt gaaacaccat ttctactaaa actacaaaaa attagctggg 120catggtggcg ggcgcctgta atcccagcta ctcaggaggc tgaagcagaa gaatggct 178 22180 DNA Homo sapiens /note=“Intronic region flanking exon 13, pos.48256 - 48436” 22 cctgtaaccc cagcactttg ggaggccaag gcaggcgaat cacctgaggtcgggagctcg 60 agaccagcct gaccaacatg gagaaaccac atctctacta aaactacaaaaaattagccg 120 ggcgtggtgg cacatgcctg taatcccagc tacttgggag ctacggtgcctggcctagtt 180 23 60 DNA Homo sapiens /note=“Deletion-function fragment”23 agaccatcct ggctaacacg gtgaaacacc atttctacta aaactacaaa aaattagccg 60

What is claimed is:
 1. A labeled probe for detecting a deletion of astretch of nucleotides from a BRCA1 gene, wherein said deletioncomprises exon 13 or exon 22, wherein the probe comprises nucleic acidsequences complementary to both sides of said deletion, and wherein theprobe comprises a nucleotide sequence which is the product of a fusionbetween two ALU-elements in the BRCA1 gene.
 2. A method for determiningthe presence in a sample of a nucleic acid derived from a BRCA1 genehaving a deletion of a stretch of nucleotides, wherein said deletioncomprises exon 13 or exon 22; the method comprising: (i) contacting saidsample with at least one probe which alone or together with a means fordetecting said deletion, distinguishes between a BRCA1 gene having saiddeletion and a BRCA1 gene not having said deletion, and (ii) allowinghybridization between said probe and said nucleic acid to form ahybridization product, and (iii) identifying the hybridization product.3. The method according to claim 2, wherein the probe is labeled.
 4. Themethod according to claim 2, wherein the probe comprises nucleic acidsequences complementary to both sides of the deletion.
 5. The methodaccording to claim 2, wherein the nucleic acid derived from a BRCA1 geneis amplified.
 6. The method according to claim 5, wherein the probecomprises a nucleic acid sequence which is the product of a fusionbetween two ALU-elements in the BRCA1 gene.
 7. The method according toclaim 2, wherein the hybridization product is quantified.
 8. A methodfor determining the presence in a sample of a nucleic acid derived froma BRCA1 gene having a deletion of a stretch of nucleotides, wherein saiddeletion comprises exon 13 or exon 22; the method comprising: (i)contacting said sample with a primer pair which alone or together with ameans for detecting said deletion, distinguishes between a BRCA1 genehaving said deletion and a BRCA1 gene not having said deletion, (ii)amplifying said sample to form an amplified product, and (iii)identifying the amplified product.
 9. The method according to claim 8,further comprising contacting the amplified product with a second primerpair for amplification, and wherein the two primer pairs comprise anested set.
 10. The method according to claim 8, wherein the primer pairis suitable for amplification by PCR or NASBA.